JP2023505506A - Methods of treating chemotherapy- or radiotherapy-induced neutropenia - Google Patents

Abstract

A method of treating chemotherapy-induced neutropenia in a patient in need thereof by administering an effective amount of eflapegrastim to the patient, comprising administering an effective amount of eflapegrastim to the patient thereby providing a method of treating radiation-induced neutropenia in a patient in need thereof.

Description

The present invention relates to pharmaceutical compositions comprising protein complexes and their pharmaceutical uses for treating and preventing conditions characterized by impaired leukopoiesis, such as neutropenia. The protein conjugate is also formed by linking an immunoglobulin Fc region to a bioactive polypeptide via a non-peptide polymer linked to the immunoglobulin Fc region.

Human granulocyte colony-stimulating factor (G-CSF) is a glycopoietic protein produced by stromal cells, macrophages, endothelial cells, fibroblasts and monocytes. The G-CSF binds with high affinity to G-CSF receptors expressed on neutrophil progenitor cells of the bone marrow, allowing neutrophil progenitor cells to proliferate without significant hematopoietic effects on other lineages of blood cells. , induces them to differentiate into neutrophils that fight infection. The use of recombinant G-CSF preparations prevents the development of severe myelosuppression and associated complications in patients with non-myelogenous malignancies undergoing radiation or chemotherapy, and febrile neutropenia (FN: It is a well-established therapy for accelerating bone marrow recovery to reduce febrile neutropenia.

Pegfilgrastim (Neulasta®; Amgen Inc.) is the most popular PEGylated form of recombinant human G-CSF. Eflapegrastim was developed to reduce the severity and duration of severe neutropenia, as well as the complications of neutropenia associated with the use of myelosuppressive anticancer agents. Continuous G-CSF. Currently, the recommended dosing regimen for both eflapegrastim (Rolontis®, HM10460A) and pegfilgrastim is dosing the next day after cytotoxic chemotherapy, which is generally followed by chemotherapy. After receiving the treatment, the weak and inconvenient patient still has to go to the hospital.

WO2019/152530

Therefore, a daily dosing regimen for sustained G-CSF that reduces patient burden while providing similar or better efficacy in the treatment of neutropenia must be developed. , there are requirements to be satisfied

In one aspect, increasing absolute neutrophil count, granulocyte count in a subject eligible for bone marrow transplantation, stem cell production, hematopoiesis, hematopoietic progenitor cell count, or stem cell production in a stem cell donor in a patient in need of such treatment and comprising administering an effective amount of eflapegrastim within a period of less than 24 hours after the patient has been administered a chemotherapeutic agent.

In another aspect, a method of treating or preventing a condition characterized by impaired leukopoiesis in a patient in need thereof, wherein less than 24 hours after the patient has been administered a chemotherapeutic agent administering an effective amount of eflapegrastim for a period of time.

In yet another embodiment, absolute neutrophil count, granulocyte count in a subject eligible for bone marrow transplantation, stem cell production, hematopoiesis, hematopoietic progenitor cell count, or stem cell in a stem cell donor in a patient in need of such treatment A method of increasing production is provided comprising administering an effective amount of eflapegrastim within a period of less than 24 hours after a patient has received radiation therapy.

In yet another aspect, a method of treating or preventing a condition characterized by impaired leukopoiesis in a patient in need thereof, wherein less than 24 hours after the patient has been administered radiation therapy administering an effective amount of eflapegrastim for a period of time.

In some embodiments, the condition characterized by impaired leukopoiesis is chemotherapy-induced neutropenia, radiation therapy-induced neutropenia, impaired hematopoietic function, impaired immune function, decreased reduced neutrophil count, reduced neutrophil recruitment, recruitment of peripheral blood progenitor cells, sepsis, bone marrow transplantation, infectious disease, leukopenia, thrombocytopenia, anemia, increased bone marrow engraftment during transplantation, selected from the group consisting of enhanced bone marrow recovery in radiotherapy, compound-induced or chemotherapeutic agent-induced bone marrow amorphia or myelosuppression, radiotherapy-induced bone marrow amorphia or myelosuppression, and acquired immune deficiency syndrome be.

In some embodiments, the condition characteristic of impaired leukopoiesis is chemotherapy-induced neutropenia or radiation therapy-induced neutropenia.

In some embodiments, the methods reduce the duration of chemotherapy-induced neutropenia or radiotherapy-induced neutropenia in a patient in need thereof.

In some embodiments, the method comprises administering an effective amount of eflapegrastim on the same day that the patient is administered chemotherapy or radiotherapy.

In some embodiments, administration of an effective amount of eflapegrastim provides a duration of absolute neutrophil count of less than about 0.5×10 ⁹ /L in the patient for less than about 6 hours, less than about 12 hours, or 24 hours. Shorten to less than

In some embodiments, administration of an effective amount of eflapegrastim prevents absolute neutrophil counts from falling below about 0.5×10 ⁹ /L in the patient.

In some embodiments, upon administration of an effective amount of eflapegrastim, the patient's absolute neutrophil count decreases from an initial occurrence of less than about 0.5×10 ⁹ //L to about 1.5×10 ⁹ /L or more, to about It can be increased within less than 4 days, less than about 7 days, or less than about 10 days.

FIG. 2 shows the results of chromatography of immunoglobulin Fc fragments obtained by papain-cleavage of immunoglobulins. FIG. 2 shows SDS-PAGE results of purified immunoglobulin Fc fragments (M: molecular size marker, lane 1: IgG, lane 2: Fc). Effect of HM10460A and Pegfilgrastim on absolute neutrophil count (ANC) after acute TC-induced neutropenia in normal SD rats (0 hours post-chemotherapy (A)). , +2 hours (B), +5 hours (C) and +24 hours (D)).

As generally described herein, the present disclosure increases absolute neutrophil count, granulocyte count, stem cell production, hematopoiesis, hematopoietic progenitor cell count, or stem cell production in a patient in need of such treatment or a method of treating or preventing a condition characterized by impaired white blood cell production, wherein the patient is administered a chemotherapeutic agent or received radiation therapy within a period of less than 24 hours and administering an effective amount of eflapegrastim.

Definitions To facilitate understanding of the present invention, a number of terms and phrases are defined below.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The abbreviations used herein have their common meaning within the chemical and biological arts. The chemical structures and formulas depicted herein are to be interpreted according to standard rules of chemical valence known in the chemical arts.

Throughout the description, compositions and kits may be described as “having,” “including,” or “comprising” specified components, or processes and methods may be described as “having,” “including,” or “including” specified steps. When stated as "comprising," there are compositions and kits of the present invention that additionally consist of or are essentially made up of the listed components. It is assumed that there are methods and methods according to the invention which do and additionally consist essentially of the recited process steps or consist of the recited process steps.

In this application, when an element or component is included in and/or selected from a recited list of elements or components, that element or component is It is to be understood that any one of may be present or that the element or component may be selected from a group made up of two or more of the listed elements or components.

Also, elements and/or features of compositions or methods described herein may be modified in various ways, either explicitly or implicitly, without departing from the spirit and scope of the invention. It should be understood that they can be combined. For example, when a particular compound is referred to, that compound is also used in various embodiments of compositions of the invention and/or methods of the invention, unless the context indicates otherwise. That is, although specific examples have been described and illustrated in this application so as to create and depict a clear and concise application, examples may vary without departing from this disclosure and invention. shall be understood to be intended to be combined with or separated from.

For example, it will be understood that all features described and depicted herein may apply to all aspects of the invention described and depicted herein.

The articles "a" and "an" are used in this disclosure to denote one or more (ie, at least one) of the grammatical object of the article, unless the context is inappropriate. For example, "element" means one element or more than one element.
The term "and/or" is used in this disclosure to mean "and" or "or" unless expressly stated otherwise.

Unless otherwise understood in context and usage, the phrase "at least one of" refers individually to each of the subject matter recited after the phrase and to each of the subject matter cited, as well as various combinations of two or more of that subject matter. shall be understood to include The phrases “and/or” in relation to three or more recited subjects should be understood to have the same meaning, unless the context comprehends otherwise.

The terms "include", "include", "include", "have", "possess", "have", "contain", "contain", "or solicit" (including grammatical equivalents thereof) generally does not exclude additional, unrecited elements or steps, for example, It should be understood as open and non-restrictive.

Where the use of the term "about" precedes a quantitative value, the invention also includes the particular quantitative value itself unless specifically stated otherwise. As used herein, the term "about" refers to ±10% variation from the stated value, unless the context indicates or infers otherwise.

At various places in this specification, variables or parameters are described as groups or ranges. The description is specifically intended to include all individual subcombinations of each of the members of such groups and ranges. For example, integers ranging from 0 to 40 are specifically , 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 and 40 individually Disclosed, integers ranging from 1 to 20 are specifically 19 and 20 separately.

For example, any and all examples, such as "such as" or "including", or the use of exemplary language are merely intended to further illustrate the invention and unless claimed , are not intended to limit the scope of the invention. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Compositions generally specifying percentages are by weight unless otherwise specified. Also, if a variable is not accompanied by a definition, the previously given definition of that variable applies.

As used herein, the term "severe neutropenia" is defined as neutropenia with an absolute neutrophil count of less than 0.5 x ¹⁰⁹ /L. The terms "severe neutropenia" and "grade 4 neutropenia" are also used interchangeably.

As used herein, "pharmaceutical composition" or "pharmaceutical dosage form" refers to a combination of an active agent and an inert or active carrier that renders the composition in vivo or in vitro. , making it particularly suitable for diagnostic and therapeutic applications.

"Pharmaceutically acceptable" means approved or approved by federal or state regulatory agencies in the United States, or by such agencies in countries other than the United States, or by the United States Pharmacopoeia or other general means those listed in a recognized pharmacopoeia for use in animals, particularly humans.

"Pharmaceutically acceptable excipient" as used herein refers to a substance that aids in the administration of and/or absorption by a subject of an active agent and does not cause significant adverse toxicological effects to the patient. It refers to a substance that can be included in the compositions of the present invention without causing arousal. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, saline solutions (eg, phosphate buffered saline solutions), emulsions (eg, oil-in-water or water-in-oil emulsions), lactate. Infusion, normal sucrose, common glucose, binders, fillers, disintegrants, lubricants, coating agents, sweeteners, flavors, salt solutions (e.g. Ringer's solution), alcohols, oils, gelatin, carbohydrates (e.g. : lactose, amylose or starch), fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine and pigments. Such formulations may be sterile and, if desired, contain lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts affecting osmotic pressure, buffers, which do not deleteriously react with the compounds of this invention. It can be mixed with adjuvants such as colorants and/or aromatic substances. For examples of such excipients, see Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA (1975).

"Subjects" contemplated for administration include humans (e.g., male or female of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adult or geriatric)), and/or non-human animals such as primates (e.g., Cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats and/or dogs. , but not limited to them. In certain embodiments, the subject is human. In certain embodiments, the subject is a non-human animal.

"Administering" as used herein includes oral administration, administration as a suppository, topical contact, intravenous administration, parenteral administration, intraperitoneal administration, intramuscular administration, intralesional administration, intraspinal administration, cranial administration, Refers to intranasal, intranasal or subcutaneous administration, or implantation of a slow release device (eg, a mini-osmotic pump) into the subject. The administration is by any route, including parenteral and transmucosal (eg, buccal, sublingual, palatal, gingival, nasal, vaginal, rectal or transdermal). Such parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intracerebroventricular and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous injections, transdermal patches, and the like. "Concurrent administration" means that the compositions described herein are administered concurrently with, immediately prior to, or immediately following administration of one or more additional therapies (e.g., anticancer agents, chemotherapy, radiation therapy, or treatment for neurodegenerative disease). Means to be administered. Eflapegrastim is administered to patients alone or in combination. The co-administration is meant to include administration of the compounds either individually or in combination (one or more compounds or agents), simultaneously or sequentially. Therefore, if desired, the formulation can be combined with other active substances (eg, to reduce metabolic degradation).

The terms "disease," "disorder," and "condition" may be used interchangeably herein.

As used herein, the terms "treat," "treating," and "treatment," unless otherwise specified, occur while a subject is suffering from a particular disease, disorder, or condition. Actions that reduce the severity of a disease, disorder, or condition, or slow or slow the progression of a disease, disorder, or condition (eg, “therapeutic treatment”) are considered.

Generally, an "effective amount" of a compound refers to an amount sufficient to elicit the desired biological response, eg, to treat upper urothelial carcinoma or non-muscle invasive bladder cancer. As one skilled in the art will appreciate, the effective amount of the compounds of the present disclosure will vary depending on the desired biological endpoint, the pharmacokinetics of the compound, the disease to be treated, the mode of administration, the age, weight, health and condition of the subject. It also depends on factors such as

The term "protein conjugate" or "slide" as used herein refers to one or more physiologically active polypeptides, one or more non-peptide polymers having reactive groups at both termini, and one or more immunoglobulin Fc Refers to a compound, including fragments, whose three components are covalently linked. It also includes only two different molecules selected from among physiologically active polypeptides, non-peptide polymers and immunoglobulin Fc fragments, to distinguish it from a "slurry", wherein the two molecules are covalently bound A constructed construct is designated a "composite".

As used herein, the term "immunoglobulin Fc fragment" includes immunoglobulin heavy chain constant region 2 (C _H 2) and heavy chain constant region 3 (C _H 3), but The variable regions, heavy chain constant region 1 (C _H 1) and light chain constant region 1 (C _L 1), refer to proteins without. It also includes the hinge region in the heavy chain constant region. In addition, the immunoglobulin Fc fragment of the present invention, except for the variable regions of the heavy and light chains, contains a part or all of heavy chain variable region 1 (C _H 1) and/or light chain constant region 1 (C _L 1). It also contains Also, the IgG Fc fragment may be a relatively long portion of the C _H 2 and/or C _H 3 amino acid sequence, so long as it has a physiological function substantially similar to or better than that of the native protein. is a fragment with a deletion in . Thus, the immunoglobulin Fc fragment of the present invention comprises: 1) C _H 1, C _H 2, C _H 3 and C _H 4 domains, 2) C _H 1 and C _H 2 domains, 3) C _H 1 4) a C _H 2 domain and a C _H 3 domain; 5) _a combination of one or more domains and an immunoglobulin hinge region (or part of a hinge region); and 6) a heavy chain constant region and It also contains dimers of each domain of the light chain constant region.

As used herein, the term "deglycosylation" refers to the enzymatic removal of the glycomoieties from the Fc fragment, and the term "aglycosylation" refers to the enzymatic removal of the Fc fragment from a prokaryotic organism, preferably E. coli. produced in a non-saccharified form by

The term "combination" as used herein means that polypeptides encoding single chain immunoglobulin Fc regions of the same origin are linked to single chain polypeptides of different origin to form dimers or multimers. means to That is, the dimer or multimer is also formed by two or more fragments selected from the group consisting of IgG1 Fc fragment, IgG2 Fc fragment, IgG3 Fc fragment and IgG4 Fc fragment herein. The term "hybrid" used means that in a single-chain immunoglobulin Fc fragment there are sequences encoding two or more immunoglobulin Fc fragments of different origins.

As used herein, "non-peptidic polymer" means a biocompatible polymer comprising two or more repeating units linked together by covalent bonds other than peptide bonds.

As used herein, the terms "bioactive polypeptide," "bioactive protein," "active polypeptide," "polypeptide drug," or "protein drug" are interchangeable in their meaning and can be used in a variety of ways. It is characterized by being in a physiologically active form exhibiting in vivo physiological functions.

Efrapegrastim, also known as Efrapegrastim Rolontis®, SPI-2012, HM10460A and ^17,65 SG-CSF, is associated with severe neutropenia and myelosuppressive anticancer drugs or A long-acting granulocyte colony-stimulating factor (G-CSF) developed to reduce the severity and duration of neutropenic complications associated with the use of radiation therapy. The efrapegrastim is composed of a recombinant human G-CSF analogue (ef-G-CSF) linked to a bifunctional polyethylene glycol linker and a recombinant fragment of the Fc region of human immunoglobulin G4 (IgG4). be. In a particular embodiment, recombinant human G-CSF analog (ef-G-CSF) differs from human G-CSF (SED ID NO: 1) by substitutions of serines at positions 17 and 65 (SED ID NO: 2). Without being bound by theory, the Fc region of human IgG4 is believed to increase the serum half-life of ef-G-CSF.

ef-G-CSF is produced by E. coli transformed to a soluble form in the periplasmic space. Alternatively, Fc fragments are produced in transformed E. coli as inclusion bodies. The ef-G-CSF and Fc fragments are independently separated and purified through successive purification steps. Purified ef-G-CSF (SEQ ID NO: 2) and Fc fragments (SEQ ID NOS: 3 and 4) are 3.4 kDa polyethylene glycol (PEG) molecules designed with reactive groups at both ends. connected through Eflapegrastim itself is a molecule obtained by attachment of a PEG linker at the N-terminus of the ef-G-CSF and Fc fragments, respectively. The G-CSF analogue is a 3.4 kDa polyethylene glycol analogue (OHCCH ₂ CH ₂ (OCH ₂ CH ₂ ) _n OCH ₂ CH _{2 )} with propylaldehyde end groups on both ends at the nitrogen atom of the N-terminal Thr residue. CHO) via reductive amination to form a covalent bond. The resulting G-CSF-PEG conjugate is then ligated via reductive amination to the N-terminal Pro at the nitrogen of recombinant Fc fragment variants produced in E. coli, resulting in a final conjugate Produces a certain efrapegrastim.

In one aspect, a method of increasing absolute neutrophil count, granulocyte count in a subject suitable for bone marrow transplantation, stem cell production, hematopoiesis, hematopoietic progenitor cell count, or stem cell production in a donor in a patient in need of such treatment. provides efrapegrastim for use in a method comprising administering an effective amount of efrapegrastim within a period of less than 24 hours after a patient has been administered a chemotherapeutic agent.

In another aspect, in a patient in need of such treatment comprising administering an effective amount of eflapegrastim within a period of less than 24 hours after the patient has been administered a chemotherapeutic agent, an attenuated Eflapegrastim for use in treating or preventing conditions characterized by leukopoiesis.

In yet another aspect, increasing absolute neutrophil count, granulocyte count in a subject suitable for bone marrow transplantation, stem cell production, hematopoiesis, hematopoietic progenitor cell count, or stem cell production in a donor in a patient in need of such treatment Eflapegrastim for use in a method comprising administering an effective amount of eflapegrastim within a period of less than 24 hours after a patient has received radiation therapy.

In yet another aspect, weakened leukocytes in a patient in need thereof comprising administering an effective amount of eflapegrastim within a period of less than 24 hours after the patient has received radiation therapy. Efrapegrastim for use in treating or preventing conditions characterized by production.

Detailed descriptions in the Chemotherapy-Induced Neutropenia Treatment Section and the Radiation Therapy-Induced Neutropenia Treatment Section below also apply to efrapegrastim described in this section.

In one aspect of the pharmaceutical composition , increasing absolute neutrophil count, granulocyte count in a subject suitable for bone marrow transplantation, stem cell production, hematopoiesis, hematopoietic progenitor cell count, or stem cell production in a donor in a patient in need of such treatment eflapegrastim for use in a method of causing the patient to administer an effective amount of eflapegrastim within a period of less than 24 hours after the patient has been administered a chemotherapeutic agent; and a pharmaceutically acceptable carrier.

In another aspect, in a patient in need of such treatment comprising administering an effective amount of eflapegrastim within a period of less than 24 hours after the patient has been administered a chemotherapeutic agent, an attenuated A pharmaceutical composition comprising eflapegrastim and a pharmaceutically acceptable carrier is provided for use in treating or preventing a condition characterized by leukopoiesis.

In one aspect, a method of increasing absolute neutrophil count, granulocyte count in a subject suitable for bone marrow transplantation, stem cell production, hematopoiesis, hematopoietic progenitor cell count, or stem cell production in a donor in a patient in need of such treatment. , eflapegrastim, and a pharmaceutically acceptable A pharmaceutical composition is provided that includes a carrier.

In yet another aspect, weakened leukocytes in a patient in need thereof comprising administering an effective amount of eflapegrastim within a period of less than 24 hours after the patient has received radiation therapy. A pharmaceutical composition comprising eflapegrastim and a pharmaceutically acceptable carrier is provided for use in treating or preventing a condition characterized by production.

In certain embodiments, the pharmaceutically acceptable carrier is phosphate-buffered saline. In some embodiments, the phosphate buffered saline is Dulbecco's phosphate buffered saline. In certain embodiments, the pharmaceutically acceptable carrier is citrate buffer.

The pharmaceutical compositions provided herein can be administered orally (enteral), parenterally (by injection), rectal, transdermal, intradermal, intrathecal, subcutaneous (SC), intravenous ( It is also administered by a variety of routes including, but not limited to, IV) administration, intramuscular (IM) administration and intranasal administration. In some embodiments, the pharmaceutical compositions disclosed herein are administered parenterally. In some embodiments, the pharmaceutical compositions disclosed herein are administered by subcutaneous administration.

The pharmaceutical compositions provided herein are presented in unit dosage forms to facilitate accurate dosing. The term "dosage unit form" refers to a physically discrete unit suitable for a single dosage route for human subjects and other mammals, each unit containing the desired therapeutic effect in association with suitable pharmaceutical excipients. It contains a predetermined amount of active substance that is calculated to produce. Typical unit dosage forms include pre-filled, pre-measured ampoules or syringes of the liquid compositions or pills, tablets, capsules or the like in the case of solid compositions.

In certain embodiments, the restricted compositions provided herein are administered to the patient as subcutaneous solutions.

In certain embodiments, compounds provided herein are administered as the sole active agent or in combination with other active agents.

Although the description of pharmaceutical compositions provided herein relates primarily to pharmaceutical compositions suitable for administration to humans, such compositions are generally useful in animals of all species. It will be understood by those skilled in the art that it is suitable for administration to Modifications of pharmaceutical compositions suitable for administration to humans are well known, and a veterinary pharmacologist of ordinary skill will make such modifications in routine experimentation to provide compositions suitable for administration to a variety of animals. can be designed and/or performed. General considerations regarding the formulation and/or manufacture of pharmaceutical compositions are found, for example, in Remington: The Science and Practice of Pharmacy ^21st ed., Lippincott Williams & Wilkins, 2005.

Detailed descriptions in the Chemotherapy-Induced Neutropenia Treatment Section and Radiation Therapy-Induced Neutropenia Treatment Section below also apply to the pharmaceutical compositions described in this section.

Use and treatment
A method of increasing absolute neutrophil count, granulocyte count in a subject suitable for bone marrow transplantation, stem cell production, hematopoiesis, hematopoietic progenitor cell count, or stem cell production in a donor in one aspect of treatment of chemotherapy-induced neutropenia and wherein the patient administers an effective amount of eflapegrastim within a period of less than 24 hours after being administered the chemotherapeutic agent.

In another aspect, a method of treating or preventing a condition characterized by impaired leukocyte production in a patient in need thereof, wherein the patient is administered a chemotherapeutic agent for 24 hours. administering an effective amount of eflapegrastim for less than a period of time.

In some embodiments, the condition characterized by impaired leukopoiesis is chemotherapy-induced neutropenia, radiation therapy-induced neutropenia, hematopoietic hypofunction, immune dysfunction, neutropenia , neutrophil hypomobilization, peripheral blood progenitor cell mobilization, sepsis, bone marrow transplantation, infectious diseases, leukopenia, thrombocytopenia, anemia, increased bone marrow engraftment during transplantation, increased bone marrow recovery during radiotherapy, chemotherapy is selected from the group consisting of substance- or chemotherapy-induced myeloamorphic or myelosuppressive, radiotherapy-induced myeloamorphic or myelosuppressive, and acquired immune deficiency syndrome.

In one embodiment, the condition is chemotherapy-induced neutropenia. In one embodiment, the method can shorten the duration of chemotherapy-induced neutropenia in a patient in need thereof.

In one embodiment, the method comprises administering an effective amount of eflapegrastim on the same day that the chemotherapeutic agent is administered to the patient.

In some embodiments, administration of an effective amount of eflapegrastim can reduce the duration of absolute neutrophil counts of less than about 0.5×10 ⁹ /L to less than about 24 hours in a patient. Specifically, administration of an effective amount of eflapegrastim can result in a duration of absolute neutrophil count of less than about 0.5×10 ⁹ /L in patients less than about 24 hours, less than about 12 hours, or about 8 hours. can be shortened to less than More specifically, administration of an effective amount of eflapegrastim is associated with a duration of absolute neutrophil count of less than about 0.5 x 10 ⁹ /L in the patient for about 1 hour, about 2 hours, about 3 hours, About 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours or about 24 hours. In one embodiment, administration of an effective amount of eflapegrastim reduces the duration of absolute neutrophil counts of less than about 0.5×10 ⁹ /L to less than about 24 hours in the patient. In one embodiment, administration of an effective amount of eflapegrastim reduces the duration of absolute neutrophil counts of less than about 0.5×10 ⁹ /L to less than about 12 hours in the patient. In one embodiment, administration of an effective amount of eflapegrastim reduces the duration of absolute neutrophil counts of less than about 0.5×10 ⁹ /L to less than about 8 hours in the patient.

In some embodiments, administration of an effective amount of eflapegrastim prevents the patient's absolute neutrophil count from declining below about 0.5×10 ⁹ /L.

In some embodiments, the chemotherapy-induced neutropenia is severe neutropenia with an absolute neutrophil count of less than 0.5 x 10 ⁹ /L and upon administration of an effective amount of eflapegrastim , the patient's absolute neutrophil count is greater than or equal to about 1.5 x 10 ⁹ /L within less than about 4 days, within less than about 7 days, or within less than about 10 days from initial occurrence of less than about 0.5 x 10 ⁹ /L increase to Specifically, the time for the patient's absolute neutrophil count to recover from an initial occurrence of less than about 0.5 x ¹⁰⁹ /L to an absolute neutrophil count of about 1.5 x ¹⁰⁹ /L or greater is about 10 days. less than, less than about 7 days, or less than about 4 days. In particular embodiments, chemotherapy-induced neutropenia is severe neutropenia in which the absolute neutrophil count is less than 0.5 x 10 ⁹ /L, and in patients about 0.5 x 10 ⁹ /L for absolute neutrophil counts of less than about ¹ day, less than about 2 days, less than about 3 days, less than about 4 days, about Less than 5 days, less than about 6 days, less than about 7 days, less than about 8 days, less than about 9 days, or less than about 10 days.

In some embodiments, the method is for increasing the absolute neutrophil count of a patient in need thereof, from the patient's absolute neutrophil count of less than about 0.5×10 ⁹ /L to about 1 Time to recovery to an absolute neutrophil count of .5×10 ⁹ /L or greater is less than about 10 days. Specifically, the patient's recovery time from an absolute neutrophil count of less than about 0.5×10 ⁹ /L to an absolute neutrophil count of about 1.5×10 ⁹ /L or greater is about 10 days, about 7 days, or less than about 4 days. In particular embodiments, the time to recover from an absolute neutrophil count of less than about 0.5×10 ⁹ /L in the patient to an absolute neutrophil count of greater than or equal to about 1.5×10 ⁹ /L in the patient is about 1 day, about Less than 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days or about 10 days.

In one embodiment, an effective amount of eflapegrastim is administered concurrently with a chemotherapeutic agent.

In certain embodiments, an effective amount of eflapegrastim is administered within about 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours after administration of the chemotherapeutic agent. within, within 7 hours, within 8 hours, within 9 hours, within 10 hours, within 11 hours, within 12 hours, within 13 hours, within 14 hours, within 15 hours, within 16 hours, within 17 hours, within 18 hours, Administer within 19 hours, 20 hours, 21 hours, 22 hours, 23 hours or 24 hours.

In certain embodiments, an effective amount of eflapegrastim is administered within about 0.5 hours, within about 1 hour, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours after administration of the chemotherapeutic agent. Within hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, or within about 12 hours.

In certain embodiments, an effective amount of eflapegrastim is administered within about 0.5 hours, within about 3 hours, or within about 5 hours after administration of the chemotherapeutic agent.

In one embodiment, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent.

In certain embodiments, the myelosuppressive chemotherapeutic agent is selected from the group consisting of docetaxel, cyclophosphamide, doxorubicin, etoposide, cisplatin, paclitaxel, topotecan, vincristine, methylprednisolone, cytarabine, and combinations thereof. .

In particular embodiments, the patient is treated for a cancer selected from the group consisting of breast cancer, non-small cell lung cancer, small cell lung cancer, ovarian cancer, sarcoma, urothelial carcinoma, germ cell tumors, and non-Hodgkin's lymphoma. have been given chemotherapeutic agents for

In some embodiments, the effective amount of eflapegrastim administration comprises parenterally administering to the patient an eflapegrastim dose of about 2 mg to 18 mg. In one embodiment, the dosage is also about 13.2 mg of efrapegrastim per day.

In particular embodiments, the effective amount of eflapegrastim administered is from about 2.0 mg to about 5.0 mg, from about 5.0 mg to about 15.0 mg, from about 7.0 mg to about 15.0 mg, from about 9.0 mg to about 15.0 mg, about 11.0 mg to about 15.0 mg, about 13.0 mg to about 15.0 mg, about 5.0 mg to about 13.0 mg, about 5.0 mg to about 11.0 mg, about 5.0 mg to about 9.0 mg, about 5.0 mg to about 7.0 mg, about 7.0 mg to about 13.0 mg, about 7.0 mg to about 11.0 mg, about 7.0 mg to about 9.0 mg, about 9.0 mg to about 13.0 mg, about 9.0 mg to about 11.0 mg, about 11.0 mg to about 13.0 mg, or about 15.0 to about 18.0 mg. including parenterally administering a dose of efrapegrastim mg.

In particular embodiments, an effective amount of eflapegrastim administered is about 12.0 mg, about 12.2 mg, about 12.4 mg, about 12.6 mg, about 12.8 mg, about 13.0 mg, about 13.2 mg, parenterally administering about 13.4 mg, about 13.6 mg, about 13.6 mg, 13.8 mg, or about 14.0 mg of eflapegrastim. In certain embodiments, the effective amount of eflapegrastim administration comprises parenterally administering about 13.2 mg of eflapegrastim.

Specifically, the dose of eflapegrastim is administered within 24 hours after administration of the chemotherapeutic agent, optionally in a single dose on the day of administration of the chemotherapeutic agent to the patient, or once It may be administered in divided doses of up to 5 doses.

In one aspect of treatment of radiation therapy-induced neutropenia , absolute neutrophil count, granulocyte count in a subject eligible for bone marrow transplantation, stem cell production, hematopoiesis, hematopoietic progenitor cell count, or stem cell production in a stem cell donor and comprising administering an effective amount of eflapegrastim within a period of less than 24 hours after the patient has received radiation therapy.

In another aspect, a method of treating or preventing a condition characterized by impaired leukopoiesis in a patient in need thereof, wherein the patient has undergone radiation therapy less than 24 hours after receiving radiation therapy. A method is provided comprising administering an effective amount of eflapegrastim within a period of time.

In some embodiments, the condition characterized by compromised leukopoiesis is radiotherapy-induced neutropenia, decreased hematopoietic function, decreased immune function, decreased neutrophil count, decreased Neutrophil mobilization, mobilization of peripheral blood progenitor cells, sepsis, bone marrow transplantation, infectious diseases, leukopenia, thrombocytopenia, anemia, enhanced bone marrow engraftment during transplantation, enhanced bone marrow recovery during radiation therapy, radiation therapy It is selected from the group constituted by induced myeloamorphic or myelosuppressive and acquired immune deficiency syndromes.

In one embodiment, the condition is radiotherapy-induced neutropenia. In one embodiment, the method can shorten the duration of radiotherapy-induced neutropenia in a patient in need thereof.

In one embodiment, the method comprises administering an effective amount of eflapegrastim on the same day that the patient receives radiation therapy.

In certain embodiments, administration of an effective amount of eflapegrastim can reduce the duration of absolute neutrophil counts of less than about 0.5×10 ⁹ /L to less than about 24 hours in a patient. Specifically, administration of an effective amount of eflapegrastim results in a duration of absolute neutrophil counts of less than about 0.5×10 ⁹ /L in patients less than about 24 hours, less than about 12 hours, or less than about 8 hours. can be shortened to More specifically, administration of an effective amount of eflapegrastim is associated with a duration of absolute neutrophil count of less than about 0.5 x 10 ⁹ /L in the patient for about 1 hour, about 2 hours, about 3 hours, About 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours or about 24 hours. In one embodiment, administration of an effective amount of eflapegrastim reduces the duration of absolute neutrophil counts of less than about 0.5×10 ⁹ /L to less than about 24 hours in the patient. In one embodiment, administration of an effective amount of eflapegrastim reduces the duration of absolute neutrophil counts of less than about 0.5×10 ⁹ /L to less than about 12 hours in the patient. In one embodiment, administration of an effective amount of eflapegrastim reduces the duration of absolute neutrophil counts of less than about 0.5×10 ⁹ /L to less than about 8 hours in the patient.

In some embodiments, administration of an effective amount of eflapegrastim prevents the patient's absolute neutrophil count from falling below about 0.5×10 ⁹ /L.

In some embodiments, the radiation therapy-induced neutropenia is severe neutropenia with an absolute neutrophil count of less than 0.5×10 ⁹ /L and upon administration of an effective amount of efrapegrastim , within less than about 4 days, within less than about 7 days from the initial occurrence of an absolute neutrophil count in the patient of less than about 0.5 x 10 ⁹ /L to an absolute neutrophil count of about 1.5 x 10 ⁹ /L or greater or increased within less than about 10 days. Specifically, the time for the patient's absolute neutrophil count to recover from an initial occurrence of less than about 0.5 x ¹⁰⁹ /L to an absolute neutrophil count of about 1.5 x ¹⁰⁹ /L or greater is about 4 days. less than, less than about 7 days, or less than about 10 days. In particular embodiments, radiation therapy-induced neutropenia is defined as having an absolute neutrophil count of less than 0.5 x 10 ⁹ /L and from an absolute neutrophil count of less than about 0.5 x 10 ⁹ /L in a patient , the time to return to an absolute neutrophil count of greater than or equal to about 1.5×10 ⁹ /L is less than about 1 day, less than about 2 days, less than about 3 days, less than about 4 days, less than about 5 days, less than about 6 days , less than about 7 days, less than about 8 days, less than about 9 days, or less than about 10 days.

In some embodiments, the method is for increasing an absolute neutrophil count in a patient in need thereof, wherein the patient from an absolute neutrophil count of less than about 0.5×10 ⁹ /L, Recovery time to absolute neutrophil counts of about 1.5×10 ⁹ /L or greater is less than about 10 days in patients. Specifically, in the patient, the recovery time from an absolute neutrophil count of less than about 0.5 x ¹⁰⁹ /L to an absolute neutrophil count of about 1.5 x ¹⁰⁹ /L or greater is less than about 10 days, about Less than 7 days or less than about 4 days.

In particular embodiments, the recovery time from an absolute neutrophil count of less than about 0.5 x 10 ⁹ /L in the patient to an absolute neutrophil count of greater than or equal to about 1.5 x 10 ⁹ /L in the patient is about 1 day. less than about 2 days, less than about 3 days, less than about 4 days, less than about 5 days, less than about 6 days, less than about 7 days, less than about 8 days, less than about 9 days, or less than about 10 days.

In one embodiment, an effective amount of eflapegrastim is administered while receiving radiation therapy.

In certain embodiments, an effective amount of eflapegrastim is administered within about 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours after receiving radiation therapy. within, within 7 hours, within 8 hours, within 9 hours, within 10 hours, within 11 hours, within 12 hours, within 13 hours, within 14 hours, within 15 hours, within 16 hours, within 17 hours, within 18 hours, Administer within 19 hours, 20 hours, 21 hours, 22 hours, 23 hours or 24 hours.

In certain embodiments, an effective amount of eflapegrastim is administered within about 0.5 hours, within about 1 hour, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours after receiving radiation therapy. Within hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, or within about 12 hours.

In certain embodiments, an effective amount of eflapegrastim is administered within about 0.5 hours, within about 3 hours, or within about 5 hours after receiving radiation therapy.

In particular embodiments, the patient is treated for a cancer selected from the group consisting of breast cancer, non-small cell lung cancer, small cell lung cancer, ovarian cancer, sarcoma, urothelial cancer, germ cell tumors and non-Hodgkin's lymphoma. undergoing radiation therapy to

In some embodiments, the effective amount of eflapegrastim administration comprises parenterally administering to the patient an eflapegrastim dose of about 2 mg to 18 mg. In one embodiment, the dosage is also about 13.2 mg of efrapegrastim per day.

In particular embodiments, the effective amount of eflapegrastim administered is from about 2.0 mg to about 5.0 mg, from about 5.0 mg to about 15.0 mg, from about 7.0 mg to about 15.0 mg, from about 9.0 mg to about 15.0 mg, about 11.0 mg to about 15.0 mg, about 13.0 mg to about 15.0 mg, about 5.0 mg to about 13.0 mg, about 5.0 mg to about 11.0 mg, about 5.0 mg to about 9.0 mg, about 5.0 mg to about 7.0 mg, about 7.0 mg to about 13.0 mg, about 7.0 mg to about 11.0 mg, about 7.0 mg to about 9.0 mg, about 9.0 mg to parenterally administering a dose of about 13.0 mg, about 9.0 mg to about 11.0 mg, about 11.0 mg to about 13.0 mg, or about 15.0 mg to about 18.0 mg of eflapegrastim; including.

In particular embodiments, an effective amount of eflapegrastim administered is about 12.0 mg, about 12.2 mg, about 12.4 mg, about 12.6 mg, about 12.8 mg, about 13.0 mg, about 13.2 mg, parenterally administering about 13.4 mg, about 13.6 mg, about 13.6 mg, 13.8 mg, or about 14.0 mg of eflapegrastim. In certain embodiments, the effective amount of eflapegrastim administration comprises parenterally administering about 13.2 mg of eflapegrastim.

Specifically, the dose of eflapegrastim is administered within 24 hours of the day of radiotherapy, optionally once or in 1-5 divided doses on the day the patient received radiotherapy. can be administered.

In order that the disclosure contained herein be more fully understood, the following examples are presented. The synthetic and biological examples described in this application are provided to illustrate the compounds, pharmaceutical compositions and methods provided herein and are not to be construed as limiting their scope in any way. not to be

Example 1: Preparation of Eflapegrastim ( ^17,65 SG-CSF-PEG-Fc)
Step 1: Preparation of immunoglobulin Fc fragment using immunoglobulin The immunoglobulin Fc fragment was prepared as follows.

200 mg of immunoglobulin G (IgG, green cross) with a molecular weight of 150 kDa dissolved in 10 mM phosphate buffer was treated with 2 mg of proteolytic enzyme papain (Sigma), and reacted at 37° C. for 2 hours with gentle stirring. let me

After the enzymatic reaction, Superdex column, protein A column and cation exchange resin column chromatography were sequentially performed to purify the produced immunoglobulin Fc fragment. Specifically, the reaction solution was loaded onto a Superdex 200 column (Superdex 200, Pharmacia) equilibrated with 10 mM sodium phosphate buffer (PBS, pH 7.3), and the same buffer was used for the column. and eluted at a flow rate of 1 ml/min. Unreacted immunoglobulin molecules (IgG) and F(ab')2, which have relatively higher molecular weights than the immunoglobulin Fc fragment, were eluted earlier than the immunoglobulin Fc fragment and were first removed. Immunoglobulin Fc fragments and Fab fragments with similar molecular weights were removed by Protein A column chromatography (Fig. 1). After loading the immunoglobulin Fc fragment-containing fraction eluted with the Superdex 200 column onto a protein A column (Pharmacia) equilibrated with 20 mM phosphate buffer (pH 7.0) at a flow rate of 5 ml/min, The column was washed with the same buffer to remove proteins not bound to the column. Thereafter, the protein A column was eluted with 100 mM sodium citrate buffer (Na citrate, pH 3.0) to obtain a highly pure immunoglobulin Fc fragment. The purified Fc fraction was finally purified using a cation exchange resin column (poly CAT, Poly LC), and 10 mM acetate buffer was added to the column loaded with the Fc fraction. A highly pure immunoglobulin Fc fraction was obtained by eluting with a linear gradient of 0.15 M-0.4 M sodium chloride in pH 4.5. The highly pure immunoglobulin Fc fraction was analyzed by 10% SDS-PAGE (lane 2 in Figure 2).

Step 2: Preparation of ^17,65 SG-CSF-PEG Conjugate A 3.4-kDa polyethylene glycol with aldehyde reactive groups at both ends, ALD-PEG-ALD (Shearwater), was added to 100 mM phosphate buffer. , human granulocyte colony-stimulating factor ( ^17,65 SG-CSF, MW: 18.6 kDa) dissolved in an amount of 5 mg/ml, and ^17,65 SG-CSF:PEG molar ratio 1:5. bottom. To the mixture, the reducing agent sodium cyanoborohydride (NaCNBH ₃ , Sigma) was added to a final concentration of 20 mM and allowed to react with gentle stirring at 4° C. for 3 hours until PEG was ^17,65 SG. - was intended to be linked to the amino terminus of CSF. To obtain a 1:1 conjugate of PEG and ^17,65 SG-CSF, the reaction mixture was subjected to size exclusion chromatography using a Superdex® column (Pharmacia). The ^17,65 SG-CSF-PEG conjugate was eluted using 10 mM potassium phosphate buffer (pH 6.0) as the elution buffer, and ^17,65 SG- CSF, unreacted PEG, and the dimer by-product where PEG was linked to the ^17,65 SG-CSF molecule were removed. Purified ^17,65 SG-CSF-PEG conjugate was concentrated at 5 mg/ml. Through this experiment, the optimal reaction molar ratio of ^17,65 SG-CSF to PEG that provides the highest reactivity and produces the least amount of side products such as dimers is 1:5. was revealed.

Step 3: Preparation of 17,65S-G-CSF-PEG-Fc Conjugation To ligate the ^17,65 SG-CSF-PEG conjugate purified in Step 2 above to the N-terminus of the immunoglobulin Fc fragment Second, the immunoglobulin Fc fragment (approximately 53 kDa) produced in step 1 was dissolved in 10 mM phosphate buffer and 1:1, 1:2, 1:4 and 1:8 ^17,65 SG-CSF- The PEG conjugate:Fc molar ratio was mixed with the ^17,65 SG-CSF-PEG conjugate. After adjusting the phosphate buffer concentration of the reaction solution to 100 mM, the reducing agent NaCNBH ₃ was added to the reaction solution at a final concentration of 20 mM, and the mixture was reacted at 4° C. for 20 hours with soft stirring. Through this experiment, the optimal reaction molar ratio of ^17,65 SG-CSF-PEG conjugate to Fc that provides the highest reactivity and produces the least side products such as dimers is 1:2. It became clear.

Step 4: Separation and purification of G-CSF-PEG-Fc conjugate After the reaction in step 3, the reaction mixture was subjected to Superdex size exclusion chromatography to remove unreacted substances and by-products, resulting in ^{17, The 65} SG-CSF-PEG-Fc protein conjugate was purified. After concentrating the reaction mixture and loading it onto a Superdex column, 10 mM phosphate buffer (pH 7.3) was passed through the column at a flow rate of 2.5 ml/min to remove unbound Fc and unreacted material. After clearing, the ^17,65 SG-CSF-PEG-Fc protein conjugate fraction was collected by column elution. The collected ^17,65 SG-CSF-PEG-Fc protein conjugate fraction contains minor impurities, unreacted Fc, and interferon alpha dimer, and is subjected to cation exchange chromatography. and remove impurities. The ^17,65 SG-CSF-PEG-Fc protein conjugate fraction was loaded onto a poly CATLP column (poly LC) equilibrated with 10 mM sodium acetate (pH 4.5), and 1 M NaCl was added to the column. was used and eluted with a linear gradient of 0-0.5 M NaCl in 10 mM sodium acetate buffer (pH 4.5). Finally, the ^17,65 SG-CSF-PEG-Fc protein conjugate was purified using an anion exchange column. After loading the ^17,65 SG-CSF-PEG-Fc protein conjugate fraction onto a poly WAXLP column (poly LC) equilibrated with 10 mM Tris-HCl (pH 7.5), The highly pure form of the ^17,65 SG-CSF-PEG-Fc protein conjugate was separated using 1 M NaCl and eluted with a 0-0.3 M NaCl linear gradient in 10 mM Tris-HCl (pH 7.5). let me

Example 2 Efficacy Study of Eflapegrastim with Different Dosing Dosages in Rats with Docetaxel/Cyclophosphamide-Induced Neutropenia Eflapegrastim (HM10460A), a Long-Acting G-CSF Analogue was compared with Pegfilgrastim in a chemotherapy-induced neutropenic rat model with various dosing regimens.

In the following studies, eflapegrastim was prepared essentially as described in Example 1.

(i) research material;

Preparation of HM10460A solution for subcutaneous administration Preparation of 61.8 μg/kg HM10460A solution for subcutaneous administration: HM10460A (6.0 mg/mL) 92.7 μL stock solution was diluted with DPBS 17907.3 μL.

Preparation of 372.0 μg/kg HM10460A solution for subcutaneous administration: HM10460A (6.0 mg/mL) stock solution of 558.0 μL was diluted with DPBS 17442.0 μL.

Preparation of 496.0 μg/kg HM10460A solution for subcutaneous administration: HM10460A (6.0 mg/mL) stock solution of 744.0 μL was diluted with DPBS 17256.0 μL.

Test articles were prepared based on the G-CSF protein dosage on the pharmaceutical label (HM10460A).

HM10460A solutions for subcutaneous administration were diluted with DPBS to a final dose concentration of 2 mL/kg.

Preparation of Pegfilgrastim Solution for Subcutaneous Administration Preparation of 103.3 μg/kg Pegfilgrastim Solution for Subcutaneous Administration: Pegfilgrastim (10 mg/mL) 93.0 μL stock solution in DPBS 17907. Diluted at 0 μL.

Preparation of 620.0 μg/kg pegfilgrastim solution for subcutaneous administration: 558.0 μL of pegfilgrastim (10 mg/mL) stock solution was diluted with 17442.0 μL of DPBS.

Pegfilgrastim solutions for subcutaneous administration were diluted with DPBS to a final dose concentration of 2 mL/kg.

Preparation of neutropenia-inducing drug solution To induce neutropenia in rats, docetaxel/cyclophosphamide was administered at 1/3 human equivalent dose (docetaxel 4 mg/kg and CPA 32 mg/kg) (" TC”).

Preparation of 32 mg/kg cyclophosphamide solution for subcutaneous administration: 2560.0 g of cyclophosphamide powder (CPA, Sigma, USA) was diluted with 80000.0 μL of distilled water (DW) (Daihan, Republic of Korea).

Preparation of 4 mg/kg docetaxel solution for subcutaneous administration: Docel inj. (Hanmi Yaku, Korea) (42.68 mg/mL) 29070.0 μL was diluted with 30930.0 μL of commercial formulation buffer (FB) (127.4 mg/mL ethanol in distilled water (DW)).

Docetaxel and cyclophosphamide solutions for subcutaneous administration were diluted in FB to give a final dose concentration of 1 mL/kg. HM10460A and pegfilgrastim were diluted in DPBS to a final dose concentration of 2 mL/kg.

(ii) method
test system

Animal management and identification

animal breeding

Dosing

Group design and dose levels

(iii) observations and measurements;
Body weight Body weight was measured twice on days -1 and 0 prior to administration of TC and test substance in order to calculate appropriate volumetric doses.

ANC Profile Blood from all animals was collected from the jugular vein on day -1 pre-chemotherapy and analyzed for neutrophil count (NEUT#). The neutrophil count was used as the NEUT on Day 0 prior to dosing and grouping was based on NEUT on Day 0. Blood was also collected with a 26G 1 mL syringe at 6 hours on day 0 and once daily for 8 days after administration of the test substance. A total of 0.2 mL of blood was collected and placed in an automated hematosphere analyzer Sysmex, XN1000-V (Sysmex corp., Japan) to confirm ANC. ANC is generally calculated from Total WBC x (%Segs + %Bands), where ANC is the amount of neutrophils measured by the Sysmex system already includes neutrophil banding type in the data. is also calculated using the Sysmex system.

Duration of neutropenia profile. It was determined from the duration of cytopenia (“DN”).

(iv) Results
The time course of the ANC profile neutrophil count is illustrated in FIG. Neutrophil counts at 1/6 of the clinical dose (103.3 μg/kg pegfilgrastim and 61.8 μg/kg HM10460A) were 8 and 5-6 days after initiation of pegfilgrastim and HM10460A drug administration, respectively. , the best results were achieved without any difference between dosing regimens. Also, neutrophil counts at clinical doses (620 μg/kg pegfilgrastim and 372 μg/kg HM10460A) peaked on days 5-8 and 6 after initiation of drug administration for pegfilgrastim and HM10460A, respectively. . In addition, peak neutrophil counts were between days 6 and 7 for the high dose of HM10460A (496 μg/kg) at all time regimens without dosing regimen change.

DN profile DN values of HM10460A and pegfilgrastim administered 24 hours after chemotherapy at 1/6 clinical dose (HM10460A 61.8 μg/kg and pegfilgrastim 103.3 μg/kg) were 0.2, respectively. days and 1.8 days (Table 9). Shorter intervals between chemotherapy and administered test substance (5 hours, 2 hours and concurrently) increased the DN of pegfilgrastim to 2.4 days. In comparison, in the case of HM10460A, only a slight increase of 0.6 days was observed.

At clinical doses (HM10460A 372 μg/kg and pegfilgrastim 620 μg/kg), the DN of HM10460A and pegfilgrastim administered 24 hours after chemotherapy was observed to be 0 and 0.2 days, respectively. (Table 10). Shortening the interval between chemotherapy and administered test substance (5 hours, 2 hours and simultaneously) increased the DN of pegfilgrastim to 1.4 days. On the other hand, HM10460A administration resulted in a modest increase in DN of 0.6 days, as observed at the 1/6 clinical dose.

High dose HM10460A (496 μg/kg) showed a similar profile (0.2 days) regardless of dosing time, except for the D0+2h regimen (Table 11).

Example 3: Docetaxel 75 mg by institutional standard of care ("SOC") in early breast cancer patients after 0.5 hours of eflapegrastim administration for humans with docetaxel/cyclophosphamide induced neutropenia 13.2 mg/0.6 mL ( ^3.6 mg G-CSF) of A fixed dose was administered subcutaneously.

Example 4: Docetaxel 75 mg/m2 IV, Cyclophosphamide 600 mg/m2 IV in early breast cancer patients after 3 hours of eflapegrastim administration for humans with docetaxel/cyclophosphamide induced neutropenia Eflapegrastim 13.2 mg/0.6 mL (3.6 mg G-CSF) fixed dose was administered subcutaneously 3 hours (±15 minutes) after the end of dosing (infusion time by institutional SOC).

Example 5: Docetaxel 75 mg/m2 IV, Cyclophosphamide 600 mg/m2 IV in early breast cancer patients after 5 hours of eflapegrastim administration for humans with docetaxel/cyclophosphamide induced neutropenia Eflapegrastim 13.2 mg/0.6 mL (3.6 mg G-CSF) fixed dose was administered subcutaneously 5 hours (±15 minutes) after the end of dosing (infusion time by institutional SOC).

Example 6: A Study of the Duration of Severe Neutropenia After Various Dosing Schedules on the Same Day of Eflapegrastim Administration in Breast Cancer Patients Receiving Docetaxel and Cyclophosphamide Grade 4 Neutrophils The duration of hypopenia (absolute neutrophil count (ANC) <0.5×10 ⁹ /L) was assessed after cycle 1 of treatment.

We also evaluated the following:

• Proportion of patients with grade 4 neutropenia (ANC < 0.5 x 10 ⁹ /L) in treatment cycle 1 • ANC ≥ 1.5 x 10 ⁹ /L in severe neutropenia in treatment cycle 1 Time to recovery to Grade 3 febrile neutropenia incidence in treatment cycle 1 (ANC <1.0 x ¹⁰⁹ /L) and 1) single temperature >38.3°C (101.0°F) or 2) one of a sustained temperature ≥ 38.0°C (100.4°F) for at least 1 hour Pharmacokinetics (PK) of Eflapegrastim in Treatment Cycle 1
• Incidence of neutropenic complications, including anti-infective use and hospitalization due to neutropenia, in patients during treatment cycle 1 • Safety Eflapegrastim treatment regimen • Peripheral blood CD34 ⁺ number 13 Using a fixed dose of 2 mg/0.6 mL (3.6 mg G-CSF), same-day administration of eflapegrastim was used in patients with early-stage breast cancer following docetaxel and cyclophosphamide (TC) with variable It was administered subcutaneously (SC) on a schedule of dosing times.

treatment cycle 1
On Day 1 of Cycle 1, fixed-dose eflapegrastim was administered at one of the following time points after TC administration, after TC administration ended. 0.5 hours (±5 minutes), 3 hours (±15 minutes) and 5 hours (±5 minutes).

Prior to TC administration, patients may receive premedication for chemotherapy prophylaxis by institutional standard of care (SOC). On Day 1 of each treatment cycle, intravenous (IV) administration of TC is as follows:
• Docetaxel 75 mg/m2 IV, infusion time according to institutional SOC;
• Cyclophosphamide 600 mg/m2 IV, infusion time according to institutional SOC;
No dose modifications of docetaxel and cyclophosphamide were allowed during Cycle 1 A maximum of 45 patients were enrolled in the study, in which a 1:1:1 ratio was used and listed above subjects were randomly assigned to one of three eflapegrastim dosing schedules.

CBC (complete blood count) and blood for pharmacokinetic (PK) analysis were taken 1 day before TC administration and 1 hour (±15 min), 3 hours (±15 min) after eflapegrastim administration. , 6 hours (±15 minutes), 8 hours (±15 minutes), 24 hours (±2 hours), 48 hours (±2 hours), 72 hours (±2 hours), 144 hours (7 days±1 day) and 192 hours (9 days ± 1 day), and Cycle 2: Blood was collected 1 day (22 days) prior to TC administration. CBC analysis was performed in a clinical laboratory.

Additional CBC Samples Only in treatment cycle 1, CBC samples were taken daily from day 4 to day 10. On day 10, if the ANC was ≤1.0 x 10 ⁹ /mL, CBC samples were taken daily until the ANC was ≥ 1.5 x 10 ⁹ /mL.

Cycle 1 peripheral blood CD34 ⁺
Peripheral blood CD34 ⁺ count samples were taken from days 2 to 10.

Cycle 1 Safety Visit Treatment Cycle 2: On Day 1 (Day 22), all mandatory assessments/evaluations will be performed prior to TC administration for Treatment Cycle 2.

Interim Safety Evaluation At each eflapegrastim dosing schedule, a safety evaluation was performed once the first 3 patients completed treatment cycle 1 of the study (9 patients total). Safety assessments included adverse reactions (AEs), ANC and white blood cell (WBC) counts, duration of severe neutropenia (DSN) and neutropenic complications (hospitalization for neutropenia, febrile neutropenia, use of anti-infectives).

After completing the safety evaluation for the first three patients on each eflapegrastim dosing schedule, if there is no safety finding on any of the three eflapegrastim dosing schedules, the patients will be randomly assigned to the other enrolled in the eflapegrastim dosing schedule. If the safety study determines that one or more eflapegrastim dosing schedules must be discontinued, all newly enrolled patients will be further randomly assigned to subsequent eflapegrastim dosing schedules. rice field.

Discontinuation rule safety was assessed in the first 3 patients at each eflapegrastim dosing schedule during treatment cycle 1. Additional enrollment for an eflapegrastim schedule was discontinued if one of the following criteria was met:

1) 2 of 3 patients reported febrile neutropenia and/or all eflapegrastim-related grade 4 AEs in treatment cycle 1 2) 2 of 3 patients reported: Reported grade 4 neutropenia, DSN>2 days The safety was monitored continuously. After interim safety monitoring, the cohort was discontinued from enrollment if a total of 3 or more patients (cumulative in the cohort) exhibited febrile neutropenia (FN).

Cycle 2-4
Eflapegrastim 13.2 mg/0.6 mL (3.6 mg G-CSF) was administered within 24 hours after the end of TC administration on all eflapegrastim dosing schedules. Patients must have an ANC ≧1.5×10 ⁹ /L and a platelet count ≧100×10 ⁹ /L to begin each subsequent chemotherapy cycle. Patients were followed for safety. Each cycle is 21 days.

In treatment cycles 2-4, blood samples for CBC are taken prior to chemotherapy, on day 1 of each treatment cycle, by SOC per cycle. The CBC was generated at the study termination visit 35 (±5) days after the last dose of study treatment (TC or eflapegrastim).

Study period Screening period: up to 30 days Treatment period: up to 4 treatment cycles (21 days per treatment cycle)
Safety Follow-up Visit for Treatment Cycle 1: In Treatment Cycle 2, 1 day prior to TC administration (day 22)
End of study visit: 35 (±5) days after the last dose of study treatment (TC or eflapegrastim)
Inclusion criteria
Patients must be willing and able to provide written consent, adhere to timed administration of efrapegrastim, adhere to blood collection schedules, and meet all other study requirements.

Patients must undergo a new diagnosis for histologically confirmed early breast cancer (ESBC), which is limited to operable stage I-IIIA breast cancer.

Patients must be candidates to receive adjuvant TC chemotherapy or neoadvant TC chemotherapy.

Patients (male or female) must be 18 years or older.

Patients must have adequate hematological, renal and hepatic function defined as follows.

● ANC≧1.5×10 ⁹ /L
●Platelet count≧100×10 ⁹ /L
● Hemoglobin ≥ 10g/dL
- Calculated creatinine clearance >50 mL/min
● Total bilirubin ≤ 1.5 mg/dL
Aspartate aminotransferase (AST)/serum glutamate oxaloacetate transaminase (SGOT and alanine aminotransferase) (ALT)/serum glutamate pyruvate transaminase (SGPT) < 2.5 x ULN and alkaline phosphatase < 2.0 x ULN
Patients must have an ECOG (eastern cooperative oncology group) performance status <2.

Eflapegrastim is supplied in pre-filled sterile single-use syringes with 13.2 mg/0.6 mL of efrapegrastim (3.6 mg G-CSF). Dose modification of efrapegrastim is not allowed.

Duration of Grade 4 neutropenia (ANC<0.5×10 ⁹ /L) was assessed in Evaluation Treatment Cycle 1.

In treatment cycle 1, the proportion of patients with grade 4 neutropenia (ANC<0.5×10 ⁹ /L) was evaluated.

In treatment cycle 1, the time to resolution of severe neutropenia to ANC ≧1.5×10 ⁹ /L was assessed.

Incidence of grade 3 febrile neutropenia (ANC < 1.0 x ¹⁰⁹ /L) and single temperature ≥ 38.3°C (101.0°F) or sustained temperature ≥ 38.3°C (101.0°F) in treatment cycle 1 One hour or more at 38.0°C (100.4°F) was rated.

The pharmacokinetics (PK) of eflapegrastim in treatment cycle 1 was evaluated.

During treatment cycle 1, patients were assessed for the incidence of neutropenic complications, including anti-infective use and hospitalization due to neutropenia.

Peripheral blood CD34 ⁺ counts were assessed.

Pharmacokinetic Evaluation Each patient received a fixed dose of efrapegrastim after starting chemotherapy on day 1, according to each eflapegrastim dosing schedule. Blood samples for pharmacokinetic measurements and CBC were collected according to the following schedule.

Cycle 1 Day 1 pre-dose (before TC dose)
1, 3, 6 and 8 hours (±15 minutes) from the time of eflapegrastim administration
24, 48 and 72 (± 2 hours) from the time of administration of eflapegrastim on day 1
144 hours (day 7 ± 1 day) and 192 hours (day 9 ± 1 day) from the time of administration of eflapegrastim on day 1
Day 1 of Cycle 2
Before TC administration
Additional CBC sample
Only in Cycle 1, CBC were also collected daily from day 4 to day 10. On day 10, if the ANC was ≤1.0 x 10 ⁹ /L, CBC was collected daily until the ANC was ≥ 1.5 x 10 ⁹ /L.

Peripheral Blood CD34+ Counts in Cycle 1 Peripheral blood CD34 ⁺ counts were taken daily from day 2 to day 10.

Safety Assessments Safety was assessed throughout the study via reported/induced AEs, laboratory assessments and physical examinations.

INCORPORATION BY REFERENCE This application references various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. In the event of a conflict between the incorporated reference and this specification, the specification will control. Also, any particular embodiment of the present disclosure that is prior art is expressly excluded from one or more of the claims. Such examples are considered well known to those skilled in the art and are hereby excluded even if the exclusion is not expressly indicated. Any particular embodiment of the present disclosure may be excluded from any claim for any reason, regardless of whether or not it relates to the existence of prior art.

Equivalents The invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Accordingly, the foregoing specific examples should be considered illustrative in all respects, rather than limiting the invention described herein. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein. .

[Sequence listing free text]
SEQ ID NO: 1
TPLGP ASSLP QSFLL KCLEQ VRKIQ GDGAA LQEKL A TYKLC HPEEL VLLGH SLGIP WAPLS SCPSQ ALQLA GCLSQ LHSGL FLYQG LLQAL EGISP ELGPT LDTLQ LDVAD FATTI WQQME ELGMA PALQP TQGAM PAFAS AFQRR AGGVL VASHL QSFLE VSYRV LRHLA QP
SEQ ID NO: 2
TPLGP ASSLP QSFLL KSLEQ VRKIQ GDGAA LQEKL CATYK LCHPE ELVLL GHSLG IPWAP LSSCSQALQ LAGCL SQLHS GLFLY QGLLQ ALEGI SPELG PTLDT LQLDV ADFAT TIWQQ MEELG MAPAL QPTQG AMPAF ASAFQ RRAGG VLVAS HLQSF LEVSY RVLRH LAQP
SEQ ID NO: 3
PSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHELLSNLGKQ
SEQ ID NO: 4
PSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHELLSNLGKQ

Claims (16)

Increasing absolute neutrophil count, granulocyte count, stem cell production, hematopoiesis, hematopoietic progenitor cell count, or stem cell production, or treating or preventing a condition characterized by impaired white blood cell production in a patient in need thereof a method,
A method comprising administering an effective amount of eflapegrastim within a period of less than 24 hours after a patient has been administered a chemotherapeutic agent or received radiation therapy. said condition characterized by impaired leukocyte production is chemotherapy-induced neutropenia, radiation therapy-induced neutropenia, hypohematopoiesis, hypoimmune function, decreased neutrophil count, decreased neutrophil recruitment , peripheral blood progenitor cell mobilization, sepsis, bone marrow transplantation, infectious diseases, leukopenia, thrombocytopenia, anemia, increased bone marrow engraftment during transplantation, increased bone marrow recovery during radiotherapy, chemical induction or chemotherapeutic agents 2. The method of claim 1, wherein the method is selected from the group consisting of induced myeloamorphic or myelosuppressive, radiotherapy-induced myeloamorphic or myelosuppressive, and acquired immune deficiency syndrome. 2. The method of claim 1, wherein the condition characterized by impaired white blood cell production is chemotherapy-induced neutropenia or radiation therapy-induced neutropenia. 4. The method of claim 3, wherein the method shortens the duration of chemotherapy-induced neutropenia or radiotherapy-induced neutropenia in a patient in need of such treatment. 2. The method of claim 1, wherein the method comprises administering an effective amount of efrapegrastim on the day the patient is to be administered a chemotherapeutic agent or undergoes radiation therapy. Administration of an effective amount of eflapegrastim reduces the duration of absolute neutrophil counts of less than about 0.5×10 ⁹ /L to less than about 6 hours, less than about 12 hours, or less than 24 hours in a patient. Item 6. The method according to any one of Items 1 to 5. 7. The method of claim 6, wherein administering an effective amount of eflapegrastim prevents the patient's absolute neutrophil count from falling below about 0.5 x 10 ^<9> /L. Within less than about 4 days, within about 7 days, or within about 10 days of the first occurrence of an absolute neutrophil count in the patient of less than about 0.5 x 10 ⁹ /L upon administration of an effective amount of eflapegrastim , to about 1.5×10 ⁹ /L or more. 9. The method of any one of claims 1-8, wherein an effective amount of eflapegrastim is administered concurrently with a chemotherapeutic agent or radiotherapy. 9. Of claims 1-8, wherein the effective amount of eflapegrastim is administered within about 0.5 hours, within about 3 hours, or within about 5 hours after administration of a chemotherapeutic agent or radiation therapy. A method according to any one of paragraphs. within about 0.5 hours, within about 1 hour, within about 2 hours, within about 3 hours, within about 4 hours, about 9. Any of claims 1-8 administered within 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours or within about 12 hours. A method according to any one of paragraphs. 12. The method of any one of claims 1-11, wherein the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. 13. The myelosuppressive chemotherapeutic agent according to claim 12, selected from the group consisting of docetaxel, cyclophosphamide, doxorubicin, etoposide, cisplatin, paclitaxel, topotecan, vincristine, methylprednisolone, cytarabine, and combinations thereof. the method of. for the patient to treat a cancer selected from the group consisting of breast cancer, non-small cell lung cancer, small cell lung cancer, ovarian cancer, sarcoma, urothelial cancer, germ cell tumor and non-Hodgkin's lymphoma; 14. The method of any one of claims 1-13, wherein the subject is receiving a therapeutic agent or radiotherapy. 15. The method of any one of claims 1-14, wherein administering an effective amount of eflapegrastim comprises administering about 2 mg to 18 mg of eflapegrastim parenterally. 16. The method of claim 15, wherein administering an effective amount of eflapegrastim comprises administering about 13.2 mg of eflapegrastim parenterally.

Images